FIELD OF THE INVENTION The present invention relates to chemically modifie mutant enzymes and a method of producing them and a method of screening them for amidase and/or esterase activity.

BACKGROUND OF THE INVENTION Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish et al., Ange. Chem., Int.

Ed. Engl., 34: 621-633 (1995)). However, the latter procedures are difficult to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.

Changing enzyme properties by chemical modification has been explore previously, with the first report being in 1966 by the groups of Bender (Polgar et al., J.

Am. Chem. Soc., 88: 3153-3514 (1966)) and Koshland (Neet et al., Proc. Natl. Acad. Sci.

USA, 56: 1606-1611 (1966)), who created a thiolsubtilisin by chemical transformation (CH20H CH2SH) of the active site serine residue of subtilisin BPN'to cystine.

Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by Wu (Wu et al., J. Am. Chem. Soc., 111: 4514-4515 (1989); Bell et al., Biochemistrv, 32: 3754-3762 (1993)) and Peterson (Peterson et al., Biochemistry,

34: 6616-6620 (1995)), and, more recently, Suckling (Suckling et al., Bioorcg. Med. Chem.

Let., 3: 531-534 (1993)).

Enzymes are now widely accepte as useful catalysts in organic synthesis.

However, natural, wild-type, enzymes can never hope to accept all structures of synthetic chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigue chemists for many years and continues to do so.

U. S. Patent No. 5,208,158 to Bech et al. ("Bech") describes chemically modifie detergent enzymes wherein one or more methionines have been mutated into cystines. The cystines are subsequently modifie in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with Cl 6 alkyl.

Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to identify one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile, and surface binding properties for use in, for example, detergents or organic synthesis. In particular, enzymes, such as subtilisins, tailored for peptide synthesis would be desirable. Enzymes useful for peptide synthesis have high esterase and low amidase activities. Generally, subtilisins do not meet these requirements and the improvement of the esterase to amidase selectivities of subtilisins would be desirable as would a rapid identification of subtilisins with improved esterase to amidase selectivities. However, previous attempts to tailor enzymes for peptide synthesis by lowering amidase activity have generally resulted in dramatic decreases in both esterase and amidase activities. Previous strategies for lowering the amidase activity include the use of water-miscible organic solvents (Barbas et al., J. Am. Chem. Soc., 110: 5162-5166 (1988); Wong et al., J. Am. Chem. Soc.,

112: 945-953 (1990); and Sears et al., Biotechnol. Prof., 12: 423-433 (1996)) and site- directe mutagenesis (Abrahamsen et al., Biochemistrv, 30: 4151-4159 (1991); Bonneau et al., J. Am. Chem. Soc., 113: 1026-1030 (1991); and Graycar et al., Ann. N. Y. Acad.

Sci., 67: 71-79 (1992)). However, while the ratios of esterase-to-amidase activities were improved by these approaches, the absolut esterase activities were lowered concomitantly. Abrahamsen et al., Biochemistry, 30: 4151-4159 (1991). Chemical modification techniques (Neet et al., Proc. Nat. Acad. Sci., 56: 1606 (1966); Polgar et al., J. Am. Chem. Soc., 88: 3153-3154 (1966); Wu et al., J. Am. Chem. Soc., 111: 4514-4515 (1989); and West et al., J. Am. Chem. Soc., 112: 5313-5320 (1990)), which permit the incorporation of unnatural amino acid moities, have also been applied to improve esterase to amidase selectivity of subtilisins. For example, chemical conversion of the catalytic triad serine (Ser221) of subtilisin to cystine (Neet et al., Proc. Nat. Acad. Sci., 56: 1606 (1966); Polgar et al., J. Am. Chem. Soc., 88: 3153-3154 (1966); and Nakatsuka et al., J. Am. Chem. Soc., 109: 3808-3810 (1987)) or to selenocysteine (Wu et al., J. Am.

Chem. Soc., 111: 4514-4515 (1989)), and methylation of the catalytic triad histidine (His57) of chymotrypsin (West et al., J. Am. Chem. Soc., 112: 5313-5320 (1990)) effected substantial improvement in esterase-to-amidase selectivities. Unfortunately however, these modifications were again accompanied by 50-to 1000-fold decreases in absolut esteraseactivity.

Further, previous attempts to identify enzymes with improved esterase to amidase selectivites have resulted in a slow process which requires large quantities of chemically modifie mutant enzymes. In particular, the kinetic constants of fully characterized chemically modifie mutant enzymes have been evaluated by the method of initial rates with a colorimetric assay. Amidase activity was followed by the release of p- nitroanilide from the tetrapeptide substrate succinylalanylalanylprolyphenylalanyl p- nitroanilide (sucAAPF-pNa). The analogous thiobenzyl ester substrate succinyl-alanine- alanine-proline-phenylalanine-thiobenzyl ester (sucAAPF-SBn) does not have a chromogenic leaving group, so detection of hydrolysis requires rection of the thiobenzyl leaving group with 5,5'-dithiobis-2,2'-nitrobenzoate (DTNB, Ellman's ragent).

However, the full characterization and kinetic evaluation of new chemically modifie mutant enzymes by this method is very material and time-consuming.

The present invention is directe to overcoming these deficiencies.

SUMMARY OF THE INVENTION The present invention relates to a method for screening chemically modifie mutant enzymes for amidase and/or esterase activity. This method inclues providing a chemically modifie mutant enzyme with one or more amino acid residues from an enzyme being replace by cystine residues, where at least some of the cystine residues are modifie by replacing thiol hydrogen in the cystine residues with a thiol side chain, contacting the chemically modifie mutant enzyme with a substrate for an amidase and/or a substrate for an esterase, and determining whether the chemically modifie mutant enzyme exhibits amidase and/or esterase activity.

Another aspect of the present invention relates to a chemically modifie mutant enzyme with one or more amino acid residues from an enzyme being replace by cystine residues, where at least some of the cystine residues are modifie by replacing thiol hydrogen in the cystine residue with a thiol side chain. The thiol side chain can be -SCH2 (p-CH3-C6H4),-SCH2 (p-OCH3-C6H4),-SCH2 (p-CF3-C6H4), or -SCH2 (2,4-diNO2-C6H3).

The present invention also relates to a method of producing a chemically modifie mutant enzyme. This method inclues providing an enzyme wherein one or more amino acids have been replace with cystine residues and replacing thiol hydrogen in at least some of the cystine residues with a thiol side chain to form the chemically modifie mutant enzyme. The thiol side chain can be-SCH2 (p-CH3-C6H4), -SCH2 (p-OCH3-C6H4),-SCH2 (p-CF3-C6H4), or-SCH2 (2,4-diNO2-C6H3).

The method for screening chemically modifie mutant enzymes for amidase and/or esterase activity of the present invention provides a rapid enzyme modification screen to explore new chemically modifie mutant enzymes ("CMMs") without the need to prepare large quantities of the new CMMs and without the material and time-consuming nature of the full characterization and kinetic evaluation of new CMMs.

The chemically modifie mutant enzymes of the present invention exhibit an increased esterase to amidase ratio as compare to wild-type enzymes and, therefore, more efficiently catalyze peptide synthesis. In addition, the

chemically modifie mutant enzymes of the present invention are useful in formulating various detergent compositions and in the preparation of animal feed.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the chemical modification of cystine mutants of subtilisin Bacillus lentus ("SBL") to generate chemically modifie mutant enzymes.

Figure 2 shows a 96-well colorimetric assay in accordance with the present invention.

Figure 3 is a three-dimensional plot of [kcat/Km esterase]/ [kcat/Km amidase] v. a-g thiol side chain v. chemically modifie mutant, showing the results obtained for the ratio of k, , t/Km for esterase/amidase activity. The reagents are set forth in Figure 1.

Figure 4 shows a comparison of the esterase/amidase specificity ratio from the rapid combinatorial assay of the present invention and the corresponding fully characterized enzymes.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for screening chemically modifie mutant enzymes for amidase and/or esterase activity. This method inclues providing a chemically modifie mutant enzyme with one or more amino acid residues from an enzyme being replace by cystine residues, where at least some of the cystine residues are modifie by replacing thiol hydrogen in the cystine residues with a thiol side chain, contacting the chemically modifie mutant enzyme with a substrate for an amidase and/or a substrate for an esterase, and determining whether the chemically modifie mutant enzyme exhibits amidase and/or esterase activity.

Preferably, the enzyme is a protase. More preferably, the enzyme is a Bacillus subtilisin. Subtilisins are alkaline serine protases that are finding increasing use in biocatalysis, particularly in chiral resolution, regioselective acylation of polyfunctional compound, peptide coupling, and glycopeptide synthesis. The latter two applications are of particular interest, because they provide an alternative to site-directed mutagenesis and chemical modification for introducing unnatural amino acids into proteins. Subtilisins

can catalyze peptide bond formation starting from an ester substrate, by first forming an acyl enzyme intermediate which then reacts with a primary amine to form the peptide product. This application thus requires high esterase activity to promote acyl enzyme formation and then low amidase activity to minimize hydrolysis of the peptide bond of the desired product. Generally, subtilisins do not meet these requirements and the identification of subtilisins with improved esterase to amidase selectivities is needed.

Also, preferably, the amino acids replace in the enzyme by cystines are selected from the group consisting of asparagine, leucine, or serine. More preferably, the amino acid to be replace is located in a subsite of the enzyme, preferably, the S l, S l', or S2 subsites. Most preferably, the amino acids to be replace are N62, L217, and S 166 where the numbered position corresponds to naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as Bacillus lentus subtilisin.

In a particularly preferred embodiment, the enzyme is a Bacillus lentus subtilisin. In another particularly preferred embodiment, the amino acid to be replace by cystine is N62, L217, or S 166 and the thiol side chain group is selected from the group consisting of: -SCH2C6H5; -SCH2(p-CH3-C6H4); <BR> <BR> <BR> <BR> -SCH20-OCH3-C6H4) ;<BR> <BR> <BR> <BR> <BR> <BR> -SCH20-COOH-C6H4); -SCH2C6F5; -SCH2tp-CF3-C6H4); and -SCH2 (2,4-diNO2-C6H3).

Preferably, for esterase activity, the ratio of chemically modifie mutant enzyme to substrate is from about 1 M: 10 M to about 1 M: 108 M. Most preferably, the ratio of chemically modifie mutant enzyme to substrate is about 1 M: 103 M.

Preferably, for amidase activity, the ratio of chemically modifie mutant enzyme to substrate is from about 1 M: 10 M to about 1 M: 101°M. Most preferably, the ratio of chemically modifie mutant enzyme to substrate is about 1 M: 6 x 103 M.

A"chemically modifie mutant enzyme"is an enzyme that has been changed by replacing an amino acid residue such as asparagine, leucine, or serine with a cystine residue and then replacing the thiol hydrogen of the cystine with a thiol side

chain (e. g.,-SCH2C6Hs,-SCH2 (p-CH3-C6H4),-SCH2 (p-OCH3-C6H4),-SCH2 (p-COOH- C6H4),-SCH2C6F5,-SCH2 (p-CF3-C6H4), and-SCH2 (2,4-diNO2-C6H3)). After modification, the properties of the enzyme, i. e., activity or substrate specificity, may be altered. Preferably, the activity of the enzyme is increased.

The term"enzyme"includes proteins that are capable of catalyzing chemical changes in other substances without being changed themselves. The enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include pullulanases, protases, cellulases, amylases, isomerases, lipases, oxidases, and reductases. The enzyme can be a wild-type or mutant protase. Wild-type protases can be isolated from, for example, Bacillus lentus or Bacillus amyloliquefaciens (also referred to as BPN'). Mutant protases can be made according to the teachings of, for example, PCT Publication Nos. WO 95/10615 and WO 91/06637, which are hereby incorporated by reference.

Several types of moities can be used to replace the thiol hydrogen of the cystine residue. These include-SCH2C6H5, -SCH2(p-CH3-C6H4), -SCH2 (p-OCH3- C6H4),-SCH2 (p-COOH-C6H4),-SCH2C6Fs,-SCH2 (p-CF3-C6H4), and-SCH2 (2,4-dingo2- C6H3)- The terms"thiol side chain group,""thiol containing group,"and"thiol side chain"are terms which are used interchangeably and include groups that are used to replace the thiol hydrogen of a cystine used to replace one of the amino acids in an enzyme. Commonly, the thiol side chain group inclues a sulfur through which the thiol side chain groups defined above are attache to the thiol sulfur of the cystine.

The binding site of an enzyme consists of a series of subsites across the surface of the enzyme. The substrate residues that correspond to the subsites are labeled P and the subsites are labeled S. By convention, the subsites are labeled S1, S2, S3, S4, S1' and S2'. A discussion of subsites can be found in Siezen et al., Protein Engineering, 4: 719-737 (1991) and Fersht, Enzyme Structure and Mechanism, 2 ed., Freeman: New York, 29-30 (1985), which are hereby incorporated by reference. The preferred subsites are S1, S'and S2.

Another aspect of the present invention relates to chemically modifie mutant enzymes with one or more amino acid residues from an enzyme being replace by cystine residues, where at least some of the cystine residues are modifie by replacing

thiol hydrogen in the cystine residue with a thiol side chain to form a chemically modifie mutant enzyme. The thiol side chain can be-SCH2 (p-CH3-C6H4), -SCH20-OCH3-C6H4),-SCH20-CF3-C6H4) v or-SCH2 (2,4-diNO2-C6H3).

The present invention also relates to a method of producing a chemically modifie mutant enzyme. This method involves providing an enzyme wherein one or more amino acids have been replace with cystine residues and replacing thiol hydrogen in at least some of the cystine residues with a thiol side chain to form the chemically modifie mutant enzyme. The thiol side chain can be-SCH2 (p-CH3-C6H4), -SCH20-OCH3-C6H4),-SCH20-CF3-C6H4), or-SCH2 (2,4-diNO2-C6H3).

The amino acid residues of the present invention can be replace with cystine residues using site-directed mutagenesis methods or other methods well known in the art. See, for example, PCT Publication No. WO 95/10615, which is hereby incorporated by reference. One method of modifying the thiol hydrogen of the cystine residue is set forth in the Examples.

The modifie enzymes of the present invention can be formulated into known powdered and liquid detergents having a pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergent cleaning compositions or additives can also include other enzymes, such as known protases, amylases, cellulases, lipases, or endoglycosidases, as well as builders and stabilizers.

The modifie enzymes of the present invention, especially subtilisins, are useful in formulating various detergent compositions. A number of known compound are suitable surfactants useful in compositions comprising the modifie enzymes of the present invention. These include nonionic, anionic, cationic, anionic, or zwitterionic detergents, as disclosed in U. S. Patent No. 4,404,128 to Anderson and U. S. Patent No.

4,261,868 to Flora et al., which are hereby incorporated by reference. A suitable detergent formulation is that described in Example 7 of U. S. Patent No. 5,204,015 to Caldwell et al., which is hereby incorporated by reference. The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the modifie enzymes of the present invention may be used for any purpose that native or wild-type enzymes are used. Thus, these modifie enzymes can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide synthesis, feed applications such as feed additives or preparation of feed additives, waste treatment,

textile applications such as the treatment of fabrics, and as fusion-cleavage enzymes in protein production. The modifie enzymes of the present invention may comprise improved wash performance in a detergent composition (as compare to the precursor).

As used herein, improved wash performance in a detergent is defined as increasing cleaning of certain enzyme-sensitive stains such as grass or blood, as determined by light reflectance evaluation after a standard wash cycle.

The addition of the modifie enzymes of the present invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range and the temperature is below the described modifie enzyme's denaturing temperature. In addition, modifie enzymes in accordance with the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

In another aspect of the present invention, the modifie enzymes are used in the preparation of an animal feed, for example, a cereal-based feed. The cereal can be at least one of wheat, barley, maize, sorghum, rye, oats, triticale, and riche. Although the cereal component of a cereal-based feed constitutes a source of protein, it is usually necessary to include sources of supplementary protein in the feed such as those derived from fish-meal, meat-meat, or vegetables. Sources of vegetable proteins include at least one of full fat soybeans, rapeseeds, canola, soybean-meal, rapeseed-meal, and canola- meal.

The inclusion of a modifie enzyme of the present invention in an animal feed can enable the crude protein value and/or digestibility and/or amino acid content and/or digestibility coefficients of the feed to be increased. This permits a reduction in the amounts of alternative protein sources and/or amino acids supplements which had previously been necessary ingredients of animal feeds.

The feed provided by the present invention may also include other enzyme supplements such as one or more of-glucanase, glucoamylase, mannanase, a-galactosidase, phytase, lipase, a-arabinofuranosidase, xylanase, oc-amylase, esterase, oxidase, oxido-reductase, and pectinase. It is particularly preferred to include a xylanase as a further enzyme supplement such as a subtilisin derived from the genus Bacillus.

Such xylanases are, for example, described in detail in PCT Patent Application No. WO 97/20920, which is hereby incorporated by reference.

Another aspect of the present invention is a method of treating a textile.

This method involves providing a chemically modifie mutant enzyme with one or more amino acid residues from an enzyme being replace by cystine residues, where at least some of the cystine residues are modifie by replacing thiol hydrogen in the cystine residues with a thiol side chain. The chemically modifie mutant enzyme is contacte with a textile under conditions effective to produce a textile resistant to certain enzyme- sensitive stains. Such enzyme sensitive stains include grass or blood. Preferably, the textile inclues a modifie enzyme. The method can be used to treat, for example, silk or wool as described in publications such as Research Disclosure 216,034, European Patent Application No. 134,267, U. S. Patent No. 4,533,359, and European Patent Application No. 344,259, which are hereby incorporated by reference.

EXAMPLES Example 1-Validation of Rapid Combinatorial Assay for the Preparation and Screening of Chemically Modifie Mutant Enzymes for Amidase and Esterase Activities In a 96-well enzyme activity assay, k, at/Km is obtained from the rate of product formation (v), using the limiting case of the Michaelis-Menten equation at low substrate concentration as an approximation (Equation 1, where [S] and [E] are the substrate and enzyme concentrations, respectively): V t (kCatIKM) [S] [E for [S] « KM (Equation 1) Enzyme stock solutions were prepared in 5 mM 4- morpholineethanesulfonic acid ("MES") with 2 mM Cal2, pH 6.5 at about 5 x 10-7M for amidase and 5 x 10-8M for esterase assays. Substrate solutions were prepared in dimethyl sulfoxide ("DMSO"). The amidase substrate sucAAPF-pNa stock was 1.6 mM, which gave 0.08 mM in the well. The esterase substrate succinyl-alanine-alanine-proline- phenylalanine-thiobenzyl ester ("sucAAPF-SBn") stock solution was 1.0 mM, which gave 0.05 mM in the well. Assays were carried out in 0.1 M tris hydroxymethylaminomethane ("Tris") pH 8.6 with 0.005% Tween. Tris buffer for the esterase assay contained 0.375 nM DTNB. This buffer had to be used immediately, as the DTNB decomposed within a few hours due to the high pH of the buffer. Berglund et al.,

Bioorg. Med. Chem. Lett., 6: 2507-2512 (1996) and Berglund et al., J. Am. Chem. Soc., 119: 5265-5266 (1997), which are hereby incorporated by reference.

A sample of each enzyme solution (#150 µL) was placed in a well in the lst, 5n, or 9"column of an enzyme loading plate. Rows A to G contained enzymes, and row H contained MES buffer. On a separate assay plate (Corning, flat bottom, 96-well), 10 IL of substrate solution and 180 pL of buffer were dispense into wells along columns to be used in a run. Columns 1-4 on the assay plate contained four replicates of the enzymes in column 1 of the loading plate; columns 5-8 contained four replicates of the enzymes in column 5 of the loading plate; and columns 9-12 contained four replicates of the enzymes in column 9 of the loading plate. Rections were initiated by transferring 10 VtL of enzyme solution from the loading plate to the assay plate with an 8-channel pipette. For amidase assays, four columns were initiated for one run. For esterase assays, two columns were initiated for one run. The time delay between addition of enzyme to the first column and onset of reading was 22-30 seconds (amidase) and 10-15 seconds (esterase). Immediately after initiation, the plate was placed on a Titertech Multiscan MCC340 reader (programmed in the kinetic mode, filter 414 nm, lag time 0.0 minutes, interval 5 seconds, with automatic background subtraction of blank row H) (Labsystems, Finland) and was read for 1.0 minute (amidase) or 30 seconds (esterase). Prolonge reading, past the nearly linear part of the progress curve (up to # 50% conversion), provided an underestimate of the rate. The output from the reader represented the average rate of change in absorbance at 414 nm min-1, measured at 5 second intervals, of the total time programmed. These data were converted to rates in Ms-1 using e414=8581 M-lcrri for p-nitroanilide e4l4-8708~Mcm~l for 3-carboxylate-4-nitrothiophenolate. Both extinction coefficients were determined on the reader using the same conditions and background subtraction as in the assay. The path length was 5 mm. The rates were correcte for active enzyme concentration, and the four replicates for each enzyme were averaged.

Table 1. kcat/KM obtained for amidase activity at pH 8. 6a

Enzyme Type of Assay kcatM (s mM-) WT cuvette b 76 + 7' (standardc) WT Cuvette (low substrate 75 WT 96-well e (low substrate) 75 5 t WT 96-well (standard) 80 17 L217C-96-well (low substrate) 52 ~ 6f SCH2C6H5 L217C-S 96-well 48 ~ 3 c CH2C6Hs (standard) L217C-96-well (low substrate) 113 18 I S(CHZ) sCH3 L217C-96-well 97 ~ 12 c S (CH2) sCH3 (standard) a All assays were done at room temperature (ca. 20°C). For assays in I mL curettes, the cell holder was 20°C.to bKinetic assay in I mL cuvettes. c complet enzyme kinetics by the method of initial rates, where kcat/KM was obtained from kcat and KM values and the errors were obtained from the curve-fitting error in kcat and Km. dMeasurement of kcat and KM using the low substrate approximation (see text). cAssay performed on microtiter plates instead of cuvettes. f Mean ~ standard error of 4 replicates.

The value of kcat/KM (amidase) obtained using the 96-well assay (Table 1) was the same as value obtained by the method of initial rates in 1 mL cuvettes at room temperature. The kcat/Km (amidase) values obtained for WT and CMMs at 25°C were 2 times higher than the values obtained at room temperature.

Normally, the cuvette assay is performed at 25°C. The kc,, t/Km value obtained in the cuvette at 20°C using a complete kinetic evaluation and the value calculated from the rate at the lowest substrate concentration (Equation 1) were the same, indicating that the low substrate approximation held for WT (Table 1).

To further validate the method, complete kinetic experiments were performed on 96-well plates by running 8 different substrate concentrations along

the rows and one enzyme/column. Values of kcat/KM obtained from these experiments were the same as values obtained with the low substrate approximation for WT and two L217C CMMs, indicating that the low substrate approximation also held for CMMs. Similarly, k,,, t/Km values for esterase obtained on the plate did not differ significantly from values obtained with the standard assay (Table 2).

Table 2. kcat/KM obtained for esterase activity at pH 8.6a Enzyme Type of Assay kcat/KM (s-1 mM-1) WT 96-well (low substrate) 3321 ~ 256 L217C Cuvette (standard) 5540 ~ 798 L217C 96-well (low substrat) 556'-1' 419 N62C Cuvette (standard) 4380 655 N62C 96-well (low substrate) 4151 ~ 198 N62C-SCH3 Cuvette (standard) 10100 ~ 1287 N62C-SCH3 96-well (low substrate 8739 ~ 765

a Notation as in Table 1.

In the esterase assay, temperature did not have a significant effect.

Example 2-Generation of an Array of New CMMs on a 96-well plate and Screening of the New CMMs for Amidase and Esterase Activity Using the Rapid Combinatorial Assay for the Preparation and Screening of Chemically Modifie Mutant Enzymes for Amidase and Esterase Activities For the small-scale preparation of new CMMs, combinations of 7 different aromatic methanethiosulfonate ("MTS") reagents (1 a-g) (Figure 1) and 3 cystine mutants (N62C, L217C and S 166C), as well as a WT control were set up on a 96-well plate as shown in the top part of Figure 2. Two replicates of each mutant were prepared with each ragent. One ragent, benzyl MTS (1a), had already been evaluated in large scale modifications, and was included for comparative purposes. The rection mixture in each well consiste of 20 µL of enzyme solution (in 5mM MES, 2 mM Cal2, pH 6.5, ca, 8 x 105 M), 40 suL of 2-

[N-cyclohexylamino] ethanesulfonic acid ("CHES") buffer (70 mM CHES, 5mM <BR> <BR> <BR> <BR> MES, 2mM Cal2, pH 9.5) and 10 pL of MTS reagent in acetonitrile (ca. I X 102 M). The blanks on the rection plate were 20tL of MES, 40 pL of CHES and 10 pL of acetonitrile.

The rections were left at room temperature and were tested for free thiol groups after 2 hours as follows. On a separate plate, 10 pL of rection mixture was added to 60 pL of DTNB-containing Tris buffer (pH 8.6) and the monitoring plate was scanned at 414 nm 15 minutes later. No difference between the WT and the modifie cystine mutants were detected, indicating that the rection was complet. To quench the rection, 10 pL of MES, at a pH of 6.5 was added to each well, and 20 pL of the mixture from each well was diluted with 980 pL of MES buffer (in Eppendorf tubes). An amidase loading plate was prepared with the diluted mixtures; an esterase loading plate was prepared with 10 pL of diluted mixture and 90 IL of MES buffer in the appropriate wells. From this point on, the assays were the same as described in Example 1. Each rection replicate was assayed in duplicate. The k,. at/Km values were calculated according to Equation 1, basing [E] on the concentration of active enzyme in the parent mutant and assuming no significant denaturation. Since little difference was observe between the two rection replicates, the 4 values for each combination were averaged in the final results.

The amidase kCatIKM value for N62C-a and S 166C-a obtained in the screen was about 2 times lower than the value obtained for the fully characterized enzymes (Table 3), consistent with the previous assay validation results in Example 1.

Table 3. Comparison of data from the screen and the corresponding fully characterized enzymes. Enzyme kcat/KMAmidaskcat/KMEsterasekcat/KMAmidaskcat/KMAmidase (s-1Mm-)b(s-1mM-1)1(s-1mM-1)2(s-1mM-1)a Screen Fully characterized Screen Fully characterized WT 121 3 178 15 3890 170 3560 ~ 540 N62C la 207 13 379 35 6510 560 6330 t 1360 1 c 86 6 93 4 5680 ~ 1000 7870 160 1d 74 ~ 11 442 i 36 7680 1410 50700 ~ 2160 ~3198~447520~71014260~5901e111 S166C la 10+ 1 20 1 1160 140 4920 1320 1c 17 ~ 1 68 2 1746 120 5798 106 ~172~32370~9111460~4001d16 1e 067~3784~755198~145~ aMean ~ standard error from 4 replicates, obtained at room temperature (see text). values and error calculated from individual kci,, and KM values obtained t 25°C.

The factor by which amidase k, at/Km values for N62C-a and S 166C-a differed from WT was the same for the screen and the characterized CMMs, indicating that the screen was giving the correct relative pattern for amidase activity. With respect to esterase, the kcat/KM values for WT and N62C-a were the same in the screen and the characterized enzyme (Table 3), consistent with validation results.

For S 166C-a, however, the screen underestimated the esterase kcat/Km value ~5-fold. Given that the amidase value for S 166C-a was not underestimated, the reason for the discrepancy could not be significant denaturation. The seven WT control treatments had the same esterase and amidase activity (and hence esterase/amidase ratio, Figure 3), indicating that the reagents themselves did not cause differential denaturation.

The screen results (Figure 4) suggested that N62c-d and e had greatly improved esterase activity as compare to WT. In the 166 family, some CMMs appeared to have very favorable esterase/amidase ratios. Members of the 217 family were intermediate with respect to esterase and esterase/amidase ratio.

To determine the reliability of these results, N62C-and S 166C-c, d, and e were prepared and characterized. The kcat/Km values from the screen and the characterized enzymes agreed within limits of error discussed above, except for both 4-carboxybenzyl (d) CMMs for which amidase and esterase were both #5-

fold underestimated in the screen (Table 3), because significant denaturation had occurred in both CMMs. The concentration of active enzyme was 4-fold lower than expected from the parent mutant. In spite of this problem, the screen clearly showed that both 4-carboxybenzyl (d) CMMs had improved esterase activity relative to the S-benzyl (a) variants (Table 3). Furthermore, the screen correctly predicted significantly improved esterase/amidase specificity ratios for N62C-and S 166C-c, d and e (Figure 4).

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.